Standardized Rat Coronary Ring Preparation and Real-Time Recording of Dynamic Tension Changes Along Vessel Diameter

This protocol introduces in detail the localization, separation, and the muscular ring fixation of rat coronary arteries. Providing a new method for the mechanisms, exploration, and the treatment of various cardiovascular diseases. The system is suitable for and the stable recording of the dynamic changes of in vitro arterial tension, reading in diameter from 60 micrometers to 10 millimeters.

We suggest that novices should first accurately understand the anatomical location of each vessel and can start with slightly larger vessel such as the thoracic artery and the mesenteric artery. After dissociating and removing the heart, begin by draining the residual blood from all the heart chambers by mildly squeezing with medical plastic forceps. Quickly place the pre-processed heart in a Petri dish containing 95%oxygen and 5%carbon dioxide saturated physiological salt solution at four degrees Celsius, having a pH value of 7.40.

To accurately identify the anatomical position of the coronary arteries, adjust the posture of the isolated heart under the light microscope according to the schematic diagram. Cut the left and right ventricular cavities along the inter ventricular septum from the root of the pulmonary artery with surgical scissors and tweezers. To dissociate the left and right coronary arteries from the myocardial tissue, dissect the right ventricle under an optical anatomic microscope to thoroughly expose the right coronary artery branch.

Then identify the position of the left coronary artery by rotating the heart tissue 45 degrees clockwise. After removing the surrounding sticky myocardial tissue, explicitly discern the pulsing left and right coronary arteries. Separate the coronary arteries in the middle immediately and completely immerse in physiological salt solution at four degree Celsius.

Acquire an arterial ring of about two millimeters by vertically cutting the detached artery with anatomical scissors to record the vascular tension under different stimuli. Prepare two two centimeter stainless steel wires and pre-soak in four degree Celsius physiological salt solution saturated with 95%oxygen and 5%carbon dioxide. Pass both the wires parallelly through the arterial ring along with the vessel's direction under an optical anatomical microscope and with wires of equal length exposed at both ends of the vascular cavity.

Fix the arterial ring with the steel wire front and back in the bath of the wire micrograph filled with bubbling physiological salt solution with 95%oxygen and 5%carbon dioxide. Rotate the horizontal screw knob for an appropriate front and rear spacing so that the two wires are horizontal and the arterial ring is in a natural state of relaxation. After installing the DMT bath on the thermostatic apparatus, open the data acquisition software to ensure that the corresponding path signal was recorded.

Set the parameters. Achieve the optimal initial tension of the arterial ring by applying a reasonable tension along the diameter of the vessel. At this point, set the displayed vascular tension value to zero.

Afterward, apply a three milli Newton pulse stimulus to the arterial ring by rotating the spiral axis of the bath. Click the LCD panel to set it to zero milli Newton and maintain for one hour. After incubation for one hour in oxygen saturated physiological salt solution buffer at 37 degree Celsius pH 7.40, set the tension value to zero milli Newton again on the tension control panel of the wire micrograph.

Perform the contractile activity of the coronary artery ring with the wire myograph technique and validate in three separate operations by stimulating with 60 millimolar of potassium ion solution for 10 minutes each. After each stimulation, flush the bath with the oxygen saturated physiological salt solution until vascular tone returns to its initial state. Add 0.3 micromolar U46619 to bath and observe for 10 minutes.

Add one micromolar apigenin and observe vascular effects. When dilation reaches a plateau, add the next concentration. Add one micromolar apigenin to 60 millimolar potassium pre-shrinking solution to observe the vascular effect.

When dilation reaches a plateau, add the next concentration. Add 28 millimolar potassium ion to the resting vascular ring. When the contraction reaches a plateau, add the next concentration.

Add 0.01 micromolar U46619 to the resting vascular ring. When the contraction reaches a plateau, add the next concentration. After an initial three milli Newton tension was applied to the arterial ring, its tension exceeded more than two milli Newton by applying 60 millimolar potassium ion concentration in parallel three times.

Thus the procedures above had resulted in an isolated coronary ring with excellent physiological activity. Cumulative potassium ion concentration, or U46619 were added to the bath of DMT620M, resulting in a concentration dependent increase in vitro vascular tone. The next concentration of potassium ion or U46619 was added when the vasoconstriction effect reached a plateau.

For isolated coronary rings constricted by potassium ion and U46619, the test drug apigenin in caused vasodilation in a surprisingly concentration dependent manner. The heart is dissected along the ventricular septum from the root of the pulmonary artery, exposing and separating two millimeters coronary muscular ring. Following this procedure, we can record vascular tension in retinal microvessels, renal function, renal artery, and in the middle cerebral artery.

A region dynamic from 60 micrometers to 10 millimeters.